We have made a detailed analysis of peptide properties important for forming class I/peptide/beta2m complexes using HLA-A2 as a model system. Preferential binding was for nonameric peptides having Leu or Met a position (P)2 and Val or leu a P9. The simplest peptide that we identified that could form HLA-A2 complexes had the sequence GLFGGGGGV, indicating that three nonglycine aa are sufficient for binding to HLA-A2. To determine whether most nonapeptides that contained Leu at P2 or Val or Leu at P9 could bind to HLA-A2, we tested the binding of nonapeptides selected from published HIV and melanoma protein sequences, and found that six of seven tested form stable HLA-A2 complexes, thus verifying the usefulness of these motifs for predicting antigenic peptides. We identified an optimal antigenic undecapeptide from the cytomegalovirus gB protein that could form stable HLA-A2 complexes that contained apparent anchor residues at P2 and P11 (sequence FIAGNSAYEYV), indicating that the spacing between anchor residues can be somewhat variable. Finally, we tested the importance of every amino acid residue in the influenza A matrix peptide 58-66 (sequence GILGFVFTLL) for binding to HLA-A2, by using Ala- substituted and Lys-substituted peptides. We found that multiple positions contributed to stable binding, including P2, P3, P5-P7, and P9. Furthermore, we found that l) Endogenous peptides form complexes that are about as stable as those formed with typical antigenic peptides. 2) Peptide exchange reactions proceed readily for complexes that have rapid beta2m dissociation rates. 3) The peptide exchange reaction does not require concomitant beta2m dissociation. 4) Distal parts of the class I molecule, which are not directly involved in peptide binding or beta2m binding, have a major impact on the stability of class l molecules. A motif specific to peptides that bind to the human class I major histocompatibility complex molecule HLA-A3 was identified by sequence analysis of HPLC fractions and showed that the majority contained Leu at position (P)2 and most contained Tyr or Lys at P9, and, Phe was common at P3. Six nonapeptides from within the influenza A nucleoprotein, matrix, and polymerase proteins, selected for synthesis based upon their possession of P2 and P9 anchor residues, were shown to bind HLA-A3 but not other class I molecules.